The anterior cruciate ligament (ACL) is the most frequently injured knee ligament, with over 100,000 reconstruction surgeries performed annually. Currently, the biological fixation of soft tissue-based grafts for ACL reconstruction poses a significant clinical challenge. Our approach to biological fixation centers on the regeneration of the anatomic insertion site between soft tissue and bone. Given the characteristic spatial variation in cell type, matrix composition and mineral content inherent at the enthesis, it is expected that interface regeneration will require multiple cell types and a stratified scaffold capable of supporting multi-tissue formation. Therefore, we have developed a biomimetic, multi-phased scaffold consisting of three distinct yet continuous phases, each designed for the formation of the ligament, fibrocartilage or bone regions found at the ACL-to-bone insertion. The objective of this proposal is to optimize multi-cell culture and scaffold design parameters for interface regeneration and multi-tissue formation. Aim 1 will test the hypothesis that osteoblast-fibroblast interactions can promote chondrocyte-mediated fibrocartilage formation on the scaffold. Aim 2 will optimize scaffold phase-specific mineral content and distribution. Aim 3 will focus on the formation of four distinct yet continuous tissue regions (ligament, non-mineralized fibrocartilage, mineralized fibrocartilage, and bone) through the tri-culture of fibroblasts, chondrocytes and osteoblasts on the stratified scaffold. Aim 4 will determine whether these biomimetic tissue regions formed in vitro can be maintained in vivo. Our effort to regenerate the anatomic fibrocartilage interface on ACL reconstruction grafts represents an innovative departure from the traditional focus on the non-physiologic fibrocartilage found within the bone tunnel. Moreover, the multi-phased scaffold design and tri-culture methods proposed here for multi-tissue formation are highly original. It is anticipated that the successful completion of our studies will facilitate the development of a new generation of integrative fixation devices, and our findings can be applied to many other conditions in which soft tissue-to-bone integration is also critical. PUBLIC HEALTH RELEVANCE: Biological fixation of soft tissue-based grafts for Anterior Cruciate Ligament (ACL) reconstruction poses a significant clinical challenge. We propose that the regeneration of the soft tissue-to-bone interface is a prerequisite for the functional integration of biological and synthetic grafts for ACL reconstruction. This project focuses on the design and optimization of a novel biomimetic scaffold for interface tissue engineering, using both in vitro and in vivo studies. Findings from the planned studies will have a significant impact on public health due to the large number of ACL reconstruction procedures performed nationally and worldwide. In addition, this project can have broad impact for the translation of tissue engineered grafts to the clinical setting, by enabling the formation of complex tissue systems through graft integration with each other as well as with the host environment.